Requirement of Fgf3 in epibranchial cartilage development. (A–D) Fluorescence in situ hybridization of fgf3 (green) or sox9a (green) in conjunction with the GFP immunohistochemistry (red) in Tg(sox10:GFP) reporter lines. (A’–D’) Green channel only with the Cbs outlined. Scale bar: 40 μm. Anterior is to the left. Dorsal is at the top. (A,A’)fgf3 expression in 3.5 mm SL wild-type zebrafish. Arrowheads indicate fgf3 expression in the middle and dorsal regions of Cb4 and the dorsal region of Cb3. (A’) * marks fgf3 expression in the adjacent regions to the ventral area of Cb3. (B–D)sox9a expression in wild types (B) and fgf3 mutants (C,D) at 108 hpf and 3.8 mm SL. Arrowheads mark sox9a expression in the dorsal areas of Cbs 2-5 in wild types (B) and at the ends of distorted Cbs in severe (C) and intermediate (D)fgf3 mutants. In wild types, sox9a expression is not seen in Cb1 (B). In severe fgf3 mutants, Cb1 is missing (C). In intermediate fgf3 mutants, Cb1 forms, but with no sox9a expression seen (D). (E–H) Craniofacial skeletal elements stained with Alcian Blue (cartilage) and Alizarin Red (bone). Arrows indicate Cbs, with black arrowheads marking Ebs. AECb4 is marked with red arrowheads. (E’–H’) Cropped images of the branchial basket corresponding to (E–H). (E,E’) In 24 out of 40 wild-type siblings, 4 Ebs on each side bud off at the dorsal end of the corresponding Cbs, along with AECb4 appearing on the posterior to Eb4 at 5.5 mm SL. The remaining 16 animals form 3 Ebs on each side, yet no Eb1. (F,F′) In all 6 5.5 mm SL fgf3 mutant siblings, Cb1s are relatively normal, with the other Cbs being fused or missing. Eb1 is not observed at the dorsal end of the corresponding Cb1, with no Ebs seen at the dorsal end of fused Cbs. (G,G’) In 81 out of 91 wild-type siblings treated with heat shock at 3.5 dpf for 3 days, 4 Ebs on each side form at the dorsal end of the corresponding Cbs at 9 dpf. The remaining 10 animals form 3 Ebs on each side, yet no Eb1. All 91 animals develop 5 Cbs with normal shapes. (H,H’) In 47 out of 105 Tg(hsp70I:DN-Fgfr1) animals treated with heat shock at 3.5 dpf for 3 days, no Ebs form at the dorsal end of the corresponding Cbs at 9 dpf. 44 animals form 2 Ebs (Ebs 1 and 2), without Ebs 3 and 4. The remaining 14 fish display 3 Ebs (Ebs 1-3 or Ebs 1, 3, and 4) but no Eb 2 or 4. In all 105 fish, 5 Cbs with normal shapes form. Cb, ceratobranchial cartilage; Eb, epibranchial cartilage; AECb4, accessory element of the ceratobranchial 4; D, dorsal; V, ventral. Anterior is to the left. n, number of animals analyzed.
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